One potential treatment for damaged cardiac tissue post myocardial infarction is the transplantation of autologous bone marrow-derived mesenchymal stem cells (MSCs) to stimulate angiogenesis, resulting in reperfusion of ischemic tissue that enables subsequent tissue regeneration. An inherent challenge for this approach is patient age, and thus cell donor age, which may result in decreased number and functionality of available stem cells. The objective of this proposal is to determine the effect of donor age on M8Cdependent angiogenesis for ischemic cardiac tissue. Donor ages of MSCs will be categorized as 30's, 40's, 50's, and 60's. The hypothesis is that increased donor age inhibits MSC-dependent angiogenesis within the challenging cellular and mechanical environment of the myocardium. To test this hypothesis, this proposal will utilize a differential approach of tissue engineering systems that progressively increases the complexity of the in vitro models to include, separately and in combination, heterotypic human cell phenotypes, soluble factor communication, direct cell-cell contact, 3D environments, and applied cyclic strain. Specific Aim 1 is to determine the effect of MSC donor age on endothelial cell (EC) processes and smooth muscle cell-like functions for angiogenesis in an indirect co-culture model. The hypothesis is that increased donor age mitigates MSC-dependent increases in EC proliferation, migration, and assembly through decreased secretion of VEGF. Antibody blocking and gene silencing will be used to focus on the mechanisms dependent on VEGF. Aim 2 is to determine the effect of MSC donor age on vessel-like structure formation within a cardio-relevant mechanical environment using a direct co-culture model. The hypothesis is that increased donor age mitigates MSC-dependent vessel stabilization and formation through decreased interaction with ECs via the notch signaling pathway. We will utilize 2- and 3-dimensional direct co-culture models and applied cyclic strain to assess the length, diameter, complexity, and stability of vessels formed. Gene silencing will be used to focus on the JAGGED1-N0TCH3 interactions between ECs and MSCs during direct cell-cell communication as the mechanism of vessel stabilization.